cc by-nc-nd During in vitro embryo production, chromosome screening is essential to prevent pregnancy losses caused by embryonic chromosome aberrations. When the chromosome screening is completed before fertilization, gametes are effectively utilized as genetic resources. The aim of this study was to investigate whether chromosome screening of gametes accompanied by fertilization would be feasible using a single mouse spermatozoon and oocyte. Metaphase II oocytes were divided into a cytoplast and a karyoplast. For genome cloning of the gametes, androgenic and gynogenic embryos were produced by microinjection of sperm into cytoplasts and parthenogenetic activation of karyoplasts, respectively. Pairs of blastomeres from androgenic and gynogenic embryos were fused electrically to produce diploid embryos, which were transferred into pseudopregnant surrogate mothers to examine fetal development. Blastomeres from androgenic and gynogenic embryos were individually treated with calyculin A—a specific inhibitor of type 1 and 2A protein phosphatases—for 2 h to induce premature chromosome condensation. Thereafter, chromosome analysis of blastomeres, reflecting the genetic constitution of individual spermatozoa and oocytes, was performed, and we confirmed that most of the androgenic and gynogenic 2-cell embryos had a haploid set of chromosomes in their sister blastomeres. The reconstructed embryos from blastomeres of androgenic and gynogenic 2-cell embryos could be implanted and develop into live fetuses, albeit at low efficiency. This study indicates that prezygotic chromosome screening and embryo production using a single pair of gametes may be practicable.